The present invention is directed to a method for detecting colon cancer utilizing an antibody specific for a glycoprotein found in colon cancer cells.
Colorectal cancer is among the leading causes of cancer-related morbidity and mortality in industrialized nations. The pathogenesis is related to hereditary influences, modified by the quantity and quality of dietary fat. In 1995, the American Cancer society estimated that 135,000 new cases of colon cancer were diagnosed; 71% were in the colon and 30% were in the rectum. Patients diagnosed at an early stage, prior to lymph-node spread, are potentially cured with surgery. At present, only 41% of patients are diagnosed at an early stage. The remaining cases frequently undergo peri-operative radiation and/or chemotherapy to attempt to control the metastatic spread of disease. Ultimately, 50% of patients thought to have undergone curative resections eventually develop recurrent disease. Unfortunately, 55,000 Americans die each year due to recurrent or metastatic colon or rectal cancer. The key to enhanced survival is early diagnosis. Colon and rectal cancers are often silent and slowly progressive. Most patients exhibit symptoms such as rectal bleeding, pain, abdominal distension or weight loss only after the disease is advanced and not surgically curable.
Over the past 25 years, early colorectal cancer detection has been based on the fecal occult blood test (FOBT) performed annually on asymptomatic individuals. Current recommendations adapted by several healthcare organizations, including the American Cancer Society, call for fecal occult blood testing beginning at age 50, repeated annually until such time as the patient would no longer benefit from screening. A positive FOBT leads to colonoscopic examination of the bowel; an expensive and invasive procedure, with a serious complication rate of one per 5,000 examinations. Only 12% of patients with heme positive stool are diagnosed with cancer or large polyps at the time of colonoscopy. Most studies show that FOBT screening does not improve cancer-related mortality or overall survival. Compliance with occult blood testing has been poor; less than 20percent of the population is offered or completes FOBT as recommended. If FOBT is properly done, the patient collects a fecal sample from three consecutive bowel movements. Samples are obtained while the patient adheres to dietary guidelines and avoids medications known to induce occult gastrointestinal bleeding. In reality, physicians frequently fail to instruct patients properly, patients frequently fail to adhere to protocol, and some patients find the task of collecting fecal samples difficult or unpleasant, hence compliance with annual occult blood testing is poor. Compounding the problem of compliance, the sensitivity and specificity of FOBT to detect colon cancer is poor. In eight prospective studies where hemoccult testing was followed by colonoscopy, only 41 of 159 cancers diagnosed were detected by FOBT, yielding a screening sensitivity of 26%. FOBT sensitivity for pre-cancerous polyps was also poor. Poor test specificity leads to unnecessary colonoscopy, adding considerable expense to colon cancer screening. In the University of Minnesota trial, a large prospective hemoccult screening study, test specificity was 90%, and positive predictive value was 2%. Only one colon cancer was found in every 50 test-triggered colonoscopies performed.
New methodology of immunological testing has potential advantages over FOBT including improved sensitivity, specificity and patient compliance. If immunological testing is more sensitive and specific than FOBT, the frequency of testing could be reduced, collection of consecutive samples would be eliminated, dietary and medication schedule modifications would be eliminated, and patient compliance would be enhanced. If colon cancer screening by immunological testing is more specific, the problem of false positive test results leading to unnecessary colonoscopic examination would be reduced leading to cost savings and improved safety. Clearly, there is a long felt need for a simple, accurate, and inexpensive screen for colon cancer.
Since the goblet cells of colorectal cancers produce glycoprotein mucin(s) that are immunologically distinguishable from normal colonic mucin (Nairn et al. Br. Med. J. 1791-1793, 1962) it is possible to detect their presence in the feces by immunological assay. Springer (Springer Science 1198-1206, 1984) reported that T antigens (Thomsen Friedenreich), and Tn antigens, precursors of MN blood group glycoproteins, are tumor associated antigens. Kurosaka et al (Kurosaka et al J. Biol. Chem. 258: 11594-11598, 1983) isolated several oligosaccharides from a rectal adenocarcinoma and reported that one of the major oligosaccharides was sialylated-Tn or STn (NeuAcxcex12-6Gal NAcxcex11-o-ser/thr). Ovine submaxillary mucin is identical in chemical structure as STn disaccharides. Kjeldsen et al. (Kjeldsen, et al. Cancer Res. 48: 2214-2220, 1988) produced TKH1 and TKH2 monoclonal antibodies that react with ovine submaxillary mucin and demonstrated that both were reactive by immunohistochemistry with adenocarcinoma of human lung, stomach, colon, breast and pancreas, but not with normal human tissue samples. Itzkowitz et al. (Itzkowitz et al. Cancer Res. 260: 8262-8271, 1989) observed expression of all the three Tn, Sialosyl-Tn and T antigens in colon cancers. He proposed that in malignancy incomplete glycolsylation and early sialylation of precursor antigen results in premature termination of the carbohydrate chain. In contrast, Podolsky (Podolsky J. Bio. Chem. 260: 15510-15515, 1985) had isolated 21 oligosaccharides from normal colonic mucin and sialylated-Tn was one of them. The non-reactivity of TKH2 antibody with normal colonic mucosa was explained by the work of Jass et al. (Jass et al. Pathology 26: 418-422, 1994) and later Ogata et al. (Ogata et al. Cancer Res. 55: 1869-1874, 1995) who reported that in the normal colon sialic acid is heavily o-acetylated and is masked, thus antibodies cannot react, while in neoplastic tissue o-acetylation is not extensive, and sialic acid is not masked. Removal of the O-acetyl groups from normal colon tissue by alkaline treatment made them reactive with TKH2 antibody.
Pant et al. did initial studies on Colon-Ovarian tumor antigen (COTA) with polyclonal antibodies produced against mucin extracted from human colon cancer tissue removed at surgery. The polyclonal antibodies were made specific by absorption with lyophilized extracts of normal human colon and other normal human tissues and CEA. The resultant antibody retained immunoreactivity towards colon cancer and mucinous ovarian tumors but did not react with normal colon tissue as seen by immunodiffusion and immunofluorescence testing (Pant et al. Tumor Biol. 5: 243-254, 1984). Furthermore, the absorbed anti-COTA antibodies immunostained several colon cancer tissues and LS174T tumor cells and other colon cancer xenografts but did not stain normal human colon sections (Pant et al. Am J. Clin. Path. 86:1-9, 1986). Pant used COTA isolated from LS174T tumor cells to produce a monoclonal antibody SP-21. This antibody gave identical immunohistochemical staining pattern as observed with absorbed polyclonal anti-COTA antibodies (Pant et al. Hybridoma 5: 129-135, 1986). In an extended immunohistochemical study with SP-21 monoclonal antibody, Dorman et al (Dorman et al. J. Clin. Path. 45: 932-933, 1992) observed that SP-21 immunostained several other human cancer tissues including ovary, stomach, breast, esophagus, prostate, pancreas and endometrium.
The relationship of COTA to STn was established by chemical analysis of purified COTA, blocking of immunoreactivity by N-acetyl neuraminic acid and loss of immunoreactivity after neuraminidase treatment. It was concluded that neuraminic acid is an essential component of COTA (Pant, et al. Journal of Tumor Marker Oncology 3:1-13, 1988). The identity of COTA to STn was further established by comparison of SP-21 with TKH1. Kordari et al. reported neuraminidase treatment and O-glycanase treatments of colon cancer tissue completely destroyed TKH1 and COTA epitope reactivity indicating that TKH 1 and COTA monoclonal antibodies recognized the NeuAcxcex12-6GalNAc disaccharide exclusively (Kordari, et al. Tumor Biol. Abs., 1990).
The present disclosure is drawn to a method for extracting glycoproteins from a fecal sample such that immunogenicity is maintained comprising the steps of:
(a) obtaining a fecal sample from an individual;
(b) shaking the fecal sample in a preservative solution;
(c) separating the solution containing the fecal sample to produce a fraction comprising glycoproteins;
(d) precipitating the glycoproteins from the fraction comprising glycoproteins; and
(e) dissolving the precipitated glycoproteins in buffer.
The method may further comprise the steps of:
(f) centrifuging the solution from step (e) to produce a pellet and a supernatant; and
(g) collecting the supernatant containing the extracted glycoproteins.
In a preferred embodiment, the fecal sample is collected in a clean vial containing preservative wherein the preservative comprises ethanol and formalin at a concentration such that bacterial growth is retarded and extraneous fecal matter is precipitated while maintaining immunogenicity of glycoproteins in the fecal sample. Preferably, the preservative comprises 25-45% ethanol with 0.025%-0.35% formalin. More preferably, the preservative comprises 40% ethanol with 0.25% formalin.
In a preferred embodiment, the solution containing the fecal sample is separated by centrifugation. More preferably, the centrifugation is at 1040-1500xc3x97g for 10-15 minutes at room temperature.
In a preferred embodiment, the glycoproteins are precipitated from the fraction comprising glycoproteins with 3 volumes of 100% ethanol with 0.1 ml of 20% sodium acetate. More preferably, the, precipitation proceeds for about 3 hours at room temperature. Preferably, the precipitated glycoproteins are dissolved in phosphate buffered saline.
The present disclosure is also drawn to a method for screening for colon cancer comprising:
(a) obtaining purified fecal glycoproteins, said glycoproteins being obtained by a method comprising:
(i) obtaining a fecal sample from an individual;
(ii) shaking the fecal sample in a preservative solution;
(iii) separating the solution containing the fecal sample to produce a fraction comprising glycoproteins;
(iv) precipitating the glycoproteins from the fraction comprising glycoproteins; and
(v) dissolving the precipitated glycoproteins in buffer; and
(b) determining the level of COTA antigen in the purified fecal glycoproteins.
In a preferred embodiment, the fecal sample is collected in a clean vial containing preservative wherein the preservative comprises ethanol and formalin at a concentration such that bacterial growth is retarded and extraneous fecal matter is precipitated while maintaining immunogenicity of glycoproteins in the fecal sample. Preferably, the preservative comprises 25-45% ethanol with 0.025%-0.35% formalin. More preferably, the preservative comprises 40% ethanol with 0.25% formalin.
In a preferred embodiment, the solution containing the fecal sample is separated by centrifugation. More preferably, the centrifugation is at 1040-1500xc3x97g for 10-15 minutes at room temperature.
In a preferred embodiment, the glycoproteins are precipitated from the fraction comprising glycoproteins with 3 volumes of 100% ethanol with 0.1 ml of 20% sodium acetate. More preferably, the precipitation proceeds for about 3 hours at room temperature. Preferably, the precipitated glycoproteins are dissolved in phosphate buffered saline.
In a preferred embodiment, the determination of the level of COTA antigen in the purified glycoproteins comprises the steps of:
(a) reacting an antibody for COTA antigen with the extracted glycoproteins to form a complex of the antibody and the COTA antigen;
(b) exposing the complex to a second antibody, wherein said second antibody is a detection agent; and
(c) determining the level of the detection agent and in turn determining the presence of COTA antigen in the fecal sample.
In one embodiment, the antibody for COTA antigen is bound to a solid surface. In an alternate embodiment, the extracted glycoproteins are bound to a solid surface. Preferably, the antibody for COTA antigen is monoclonal antibody SP-21.
The present disclosure is also drawn to a kit for screening for colon cancer comprising:
an anti-COTA capture antibody bound to a solid support;
purified human COTA antigen; and
a vial containing a preservative solution.
In one embodiment, the solid support is an ELISA plate. In an alternate embodiment, the solid support is a membrane filter. In a preferred embodiment, the kit contains the monoclonal antibody SP-21 as the antibody for COTA antigen.
In a preferred embodiment, the kit contains a preservative solution which comprises 25-45% ethanol with 0.025%-0.35% formalin. In a most preferred embodiment, the kit contains a preservative comprising 40% ethanol with 0.25% formalin.